Antioxidative stress compositions, methods of preparing and uses thereof

ABSTRACT

Antioxidative stress compositions, methods of using antioxidative stress composition and methods of preparing antioxidative stress composition compositions are described. The antioxidative stress compositions may be chemopreventive and/or immunomodulatory. Resveratrol, genistein, ellagic acid, curcumin and quercetin may be included in the antioxidative stress composition compositions individually or in any combination.

This application claims the benefit of U.S. Provisional Application No.62/391,115, filed on Apr. 20, 2016, and U.S. Provisional Application No.62/340,364, filed on May 23, 2016, both hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Oxidation is a chemical reaction that can produce free radicals and cancause oxidative stress. Mammals, including humans and their companionanimals, are adversely affected by the oxidative stress. The oxidativestress is therefore thought to be a source or a contributing factor ofmany disorders, including, e.g., aging, cancer, autoimmune disorders,heart disease, inflammatory disorders, bone disorders, bladder functiondisorders, joint health disorders, arthritis, vision, depression,anxiety, Alzheimer's disease, Parkinson's disease, dementias, diabetes,coronary artery disease, kidney disease, and viral or bacterialinfections.

In addition, since the average lifespan of a healthy non-human mammal(e.g., a canine) is shorter than the average lifespan of a healthy humanbeing, people commonly experience the loss of their companion animals¹.More than half of dogs over 10 years of age are likely to developcancer². Although treatment measures are available for canine neoplasia,these treatment measures are typically expensive and are not veryeffective. For example, once diagnosed with osteosarcoma, the mediansurvival time without treatment, with amputation alone, or withpalliative radiotherapy alone, is 4 months. With surgery andchemotherapy this is increased to only 10 months³. In addition, whilehuman therapies are often highly subsidized, the cost of treatingservice animals and pets generally falls wholly on the owners. Theexpense associated with treatment options varies, but may range from$6,000-10,000 for chemotherapy, $5,000-7,000 for radiation, and$2,500-6,000 for surgery⁴. Medical expenses of this magnitude are likelyto pose undue hardship on millions of people nationwide.

Antioxidative stress compositions may therefore be of value in enhancingthe health of mammals, including humans and non-human mammals (e.g.,canines). The results reported below show significant promise andsupport the notion of providing and perfecting the antioxidative stresscompositions, including chemopreventive compositions andimmunomodulatory compositions.

SUMMARY OF THE INVENTION

The invention is directed in part to antioxidative stress compositions.The antioxidative stress compositions are bioavailable and, after oraladministration, cross enterocytes and enter systemic blood circulationof mammals (e.g., canines). The antioxidative stress compositions areuseful in the treatment of disorders caused by or accompanied byoxidative stress and/or inflammation. The antioxidative stresscompositions may be administered, e.g., as an animal feed, as part of ananimal feed, as a biscuit, as a drug for use in humans or animals, or asa dietary or nutritional supplement.

The antioxidative stress compositions comprise at least three activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts of anyof the foregoing, and mixtures of any of the foregoing. Resveratrol maycomprise from about 1% to about 80% of the composition by weight,ellagic acid may comprise from about 0.25% to about 20% of thecompositions by weight, genistein may comprise from about 0.5% to about40% of the compositions by weight, curcumin may comprise from about 1%to about 80% of the compositions by weight; and quercetin may comprisefrom about 1% to about 80% of the compositions by weight.

The active agents in the antioxidative stress compositions generally donot adversely affect or block antioxidative properties of each other.The active agents work together to provide therapeutic effects (e.g.,inhibit H₂O₂-induced DNA strand breakage).

The antioxidative stress compositions are formulated such that theantioxidative stress compositions after oral or intravenousadministrations to mammals provide: (i) an increase in the production ofIL-10 in the mammals and/or (ii) a decrease in production of IL-2 in themammals and/or (iii) a decrease in production of IFN-γ in the mammalsand/or (iv) an increase in a number of monocytes and macrophages in themammals and/or (v) an increase in expression of CYBB, DUS, GSR, UCP2,and VIMP genes in the mammals and/or (vi) a downregulation of ATOX1,CCL5, EPX, MPV17, PRNP and SOD3 genes in the mammals and/or (vi) areduction of oxidative stress in the mammals. In certain embodiments,the antioxidative stress compositions are formulated such that theantioxidative stress compositions after oral and intravenousadministrations to mammals provide: (i) a mean serum level ofresveratrol of from about 3.86 ng/ml to about 154 μg/ml and/or (ii) amean serum level of resveratrol-3-O-glucuronide of from about 12.9 ng/mlto about 514 μg/ml and/or (iii) a mean serum level oftrans-resveratrol-4′-sulfate of from about 8.9 ng/ml to about 355 μg/mland/or (iv) a mean serum level of trans-Resveratrol-3-sulfate of fromabout 84.9 ng/ml to about 3397 μg/ml and/or (v) a mean serum level ofcis-resveratrol-3-sulfate of from about 2.7 ng/ml to about 108 μg/mland/or (vi) a mean serum level of resveratrol sulfate total of fromabout 36.7 ng/ml to about 1467 μg/ml and/or (vii) a mean serum level ofgenistein-4′-glucuronide of from about 14 μg/ml to about 14167 μg/mland/or (viii) a mean serum level of genistein-4′-glucuronide of fromabout 1.7 μg/ml to about 1700 μg/ml and/or (ix) a mean serum level ofgenistein sulfate of from about 54 μg/ml to about 53836 μg/ml and/or (x)a mean serum level of curcumin glucuronide of from about 6.25 ng/ml toabout 250 μg/ml and/or (xi) a mean serum level of quercetin of fromabout 0.2 ng/ml to about 8000 μg/ml. In certain embodiments, theantioxidative stress compositions are formulated such that the activeagents in the antioxidative stress compositions provide an additiveeffect(s) or a synergistic effect(s).

It is believed that the prior art does not teach or suggest compositionscomprising at least three active agents selected from the groupconsisting of resveratrol, genistein, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts of any of the foregoing, and mixtures ofany of the foregoing as disclosed herein, and, therefore, thetherapeutic effects of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing in acomposition comprising at least three active agents selected from thegroup consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing as disclosed herein are not predictablefrom the prior art. It is further believed that the in-vivo performanceof a composition comprising resveratrol, ellagic acid, curcumin,genistein and quercetin, and its therapeutic effects, are also notpredictable form the prior art.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising at leastthree active agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to protect DNAfrom oxidative stress damage in a mammal (e.g., a canine) in needthereof, and may be used to treat cancer, autoimmune disorders, heartdisease, inflammatory disorders, bone disorders, bladder functiondisorders, joint health disorders, arthritis, vision, depression,anxiety, Alzheimer's disease, Parkinson's disease, dementias, diabetes,coronary artery disease, kidney disease, and viral or bacterialinfections; or prolong longevity and/or healthspan of the mammal. Thetherapeutically effective amount of the combination may, e.g., be fromabout 0.25 mg to 8000 mg. In certain embodiments, the therapeuticallyeffective amount is from about 0.5 mg to about 7000 mg, from about 0.75mg to about 6000 mg, from about 1 mg to about 5000 mg, from about 2 mgto about 5000 mg, from about 3 mg to about 5000 mg, from about 4 mg toabout 5000 mg; from about 5 mg to about 5000 mg, from about 6 mg toabout 5000 mg, from about 7 mg to about 5000 mg, from about 8 mg toabout 5000 mg, from about 9 mg to about 5000 mg, from about 10 mg toabout 5000 mg, from about 12 mg to about 5000 mg, from about 14 mg toabout 5000 mg, from about 16 mg to about 5000 mg, from about 18 mg toabout 5000 mg, from about 20 mg to about 5000 mg, from about 25 mg toabout 5000 mg; from about 30 mg to about 5000 mg, from about 40 mg toabout 5000 mg, from about 40 mg to about 5000 mg, from about 50 mg toabout 5000 mg, or from about 100 mg to about 5000 mg. In certainembodiments, the therapeutically effective amount of the comination may,e.g., be from about 300 mg to about 8000 mg, from about 400 mg to about7000 mg, from about 500 mg to about 6000 mg, from about 750 mg to about5000 mg, from about 1000 mg to about 5000 mg, from about 1500 mg toabout 5000 mg. Resveratrol may comprise from about 1% to about 80% ofthe therapeutically effective amount by weight, ellagic acid maycomprise from about 0.25% to about 20% of the therapeutically effectiveamount by weight, genistein may comprise from about 0.5% to about 40% ofthe therapeutically effective amount by weight, curcumin may comprisefrom about 1% to about 80% of the therapeutically effective amount byweight; and quercetin may comprise from about 1% to about 80% of thetherapeutically effective amount by weight.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising at leastthree active agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to increase anumber of monocytes and macrophages in a mammal in need thereof.Monocytes and macrophages are the first cell types responding toneoplastic stimulus. The antioxidative stress composition may thereforebe used as a chemopreventive composition in the treatment or preventionof cancer in mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising at leastthree active agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to increaseexpression of CYBB, DUS, GSR, UCP2, and VIMP genes; and downregulateATOX1, CCL5, EPX, MPV17, PRNP and SOD3 genes in a mammal.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising at leastthree active agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to increaseproduction of IL-10, an inflammatory cytokine that decrease theproduction of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6),in a mammal. The antioxidative stress composition may therefore be usedto decrease inflammation, e.g., in the treatment of cancer, autoimmunedisorders, heart disease, inflammatory disorders, bone disorders,bladder function disorders, joint health disorders, arthritis, vision,depression, anxiety, Alzheimer's disease, Parkinson's disease,dementias, diabetes, coronary artery disease, kidney disease, and viralor bacterial infections.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising at leastthree active agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to decreaseproduction of IFN-γ in a mammal. IFN-γ regulates a number of genesrelated to inflammation and oxidative stress. The antioxidative stresscomposition may therefore be used to treat conditions associated withinflammation and oxidative stress (e.g., cancer, autoimmune disorders,heart disease, inflammatory disorders, bone disorders, joint healthdisorders, arthritis, vision, Alzheimer's disease, Parkinson's disease,dementias, diabetes, coronary artery disease, kidney disease, and viralor bacterial infections, etc.).

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising at leastthree active agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to decreaseproduction of IL-2.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising resveratroland at least two active agents selected from the group consisting ofgenistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of resveratrol of from about 3.86 ng/ml to about 154 μg/ml,based on oral administration to a group of eight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising resveratroland at least two active agents selected from the group consisting ofgenistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of resveratrol-3-O-glucuronide of from about 12.9 ng/ml toabout 514 μg/ml, based on oral administration to a group of eightmammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising resveratroland at least two active agents selected from the group consisting ofresveratrol, genistein, ellagic acid, curcumin, quercetin, prodrugs,metabolites, salts, and mixtures of any of the foregoing sufficient toprovide a mean serum level of trans-resveratrol-4′-sulfate of from about8.9 ng/ml to about 355 μg/ml, based on oral administration to a group ofeight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising resveratroland at least two active agents selected from the group consisting ofgenistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of trans-resveratrol-3-sulfate of from about 84.9 ng/ml toabout 3397 μg/ml, based on oral administration to a group of eightmammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising resveratroland at least two active agents selected from the group consisting ofgenistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of cis-Resveratrol-3-sulfate of from about 2.7 ng/ml toabout 108 μg/ml, based on oral administration to a group of eightmammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising resveratroland at least two active agents selected from the group consisting ofresveratrol, genistein, ellagic acid, curcumin, quercetin, prodrugs,metabolites, salts, and mixtures of any of the foregoing sufficient toprovide a mean serum level of resveratrol sulfate total of from about36.7 ng/ml to about 1467 μg/ml, based on oral administration to a groupof eight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising genisteinat least two active agents selected from the group consisting ofresveratrol, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of genistein-4′-glucuronide of from about 14 μg/ml to about14167 μg/ml, based on oral administration to a group of eight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising genisteinand at least two active agents selected from the group consisting ofresveratrol, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of genistein-4′-glucuronide of from about 1.7 μg/ml to about1700 μg/ml, based on oral administration to a group of eight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising genisteinand at least two active agents selected from the group consisting ofresveratrol, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of genistein sulfate of from about 54 μg/ml to about 53836μg/ml, based on oral administration to a group of eight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising curcuminand at least two active agents selected from the group consisting ofresveratrol, genistein, ellagic acid, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of curcumin glucuronide of from about 6.25 ng/ml to about250 μg/ml, based on oral administration to a group of eight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprising quercetinand at least two active agents selected from the group consisting ofresveratrol, genistein, ellagic acid, curcumin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to provide a meanserum level of quercetin of from about 0.2 ng/ml to about 8000 μg/,based on oral administration to a group of eight mammals.

In certain embodiments, an antioxidative stress composition comprises atherapeutically effective amount of a combination comprisingresveratrol, genistein, curcumin, and quercetin sufficient to provide(i) a mean serum level of resveratrol of from about 3.86 ng/ml to about154 μg/ml; (ii) a mean serum level of resveratrol-3-O-glucuronide offrom about 12.9 ng/ml to about 514 μg/ml; (iii) a mean serum level oftrans-resveratrol-4′-sulfate of from about 8.9 ng/ml to about 355 μg/ml;(iv) a mean serum level of trans-Resveratrol-3-sulfate of from about84.9 ng/ml to about 3397 μg/ml; (v) a mean serum level ofcis-resveratrol-3-sulfate of from about 2.7 ng/ml to about 108 μg/ml;(vi) a mean serum level of resveratrol sulfate total of from about 36.7ng/ml to about 1467 μg/ml; (vii) a mean serum level ofgenistein-4′-glucuronide of from about 14 μg/ml to about 14167 μg/ml;(viii) a mean serum level of genistein-4′-glucuronide of from about 1.7μg/ml to about 1700 μg/ml; (ix) a mean serum level of genistein sulfateof from about 54 μg/ml to about 53836 μg/ml; (x) a mean serum level ofcurcumin glucuronide of from about 6.25 ng/ml to about 250 μg/ml; and(xi) a mean serum level of quercetin of from about 0.2 ng/ml to about8000 μg/ml, based on oral administration to a group of eight mammals.

Administration of the antioxidative stress compositions of the invention(e.g., oral administration) to a mammal in need thereof preferablyresults in a reduction of oxidative stress in the mammal.

The antioxidative stress compositions may be administered orally orintravenously once a day, twice a day or three times a day for a timeperiod sufficient to provide a therapeutic effect (e.g., to reduce anantioxidative stress, or alleviate or reduce symptoms associated withthe diseases caused by or accompanied by the oxidative stress). Incertain embodiments, the time period sufficient to provide a therapeuticeffect is from 2 weeks to 20 years. In certain embodiments, the timeperiod is from 2 weeks to 15 years, from 2 weeks to 12 years, from 2weeks to 10 years, from 2 weeks to 8 years, from 2 weeks to 7 years,from 2 weeks to 6 years, from 2 weeks to 5 years, from 3 weeks to 5years, from 3 weeks to 4 years, from 4 weeks to 3 years, or from 3 weeksto 52 weeks.

Because oxidative stress causes damage to DNA, RNA, proteins, and othercellular components, the antioxidative stress compositions of thepresent invention may be used, e g., to prevent, delay the onset,prophylaxis of or in treatment of cancer, autoimmune disorders, heartdisease, inflammatory disorders, bone disorders, bladder functiondisorders, joint health disorders, arthritis, vision, depression,anxiety, Alzheimer's disease, Parkinson's disease, dementias, diabetes,coronary artery disease, kidney disease, viral or bacterial infections,and other disorders associated with, caused by or accompanied byoxidative stress. The antioxidative stress compositions may also be usedto enhance vibrancy or youthfulness or appearance of a mammal and/orimprove energy in a mammal. In addition, the antioxidative stresscompositions may be used to prolong longevity and/or healthspan ofmammals (e.g., canines). The antioxidative stress compositions may alsobe used in combination with conventional chemotherapeutic agents, toreduce or minimize side effects of the conventional chemotherapeuticagent(s) and/or speed up recovery from the administration ofconventional chemotherapeutic agent(s).

The antioxidative stress compositions of the present invention include,e.g., chemopreventive compositions and immunomodulatory compositions.

Generally, a chemopreventive composition according to the inventioncomprises an active agent selected from the group consisting ofresveratrol, genistein, ellagic acid, curcumin, quercetin, prodrugs,metabolites, salts of any of the foregoing, and mixtures of any of theforegoing (“active agents”). In certain embodiments, resveratrolcomprises from about 1% to about 80% of the chemopreventive compositionby weight, ellagic acid comprises from about 0.25% to about 20% of thechemopreventive composition by weight, genistein comprises from about0.5% to about 40% of the chemopreventive composition by weight, curcumincomprises from about 1% to about 80% of the chemopreventive compositionby weight; and quercetin comprises from about 1% to about 80% of thechemopreventive composition by weight. The chemopreventive compositionmay further comprise one or more additional agents selected from thegroup consisting of lycopene, α-tocopherol, L-selenomethionine, ellagicacid, indole-3-carbinol, sulforaphane, allicin, daidzein, EGCG, prodrugsof any of the foregoing, metabolites of any of the foregoing, and saltsof any of the foregoing. The chemopreventive composition may furthercomprise one or more additional antioxidant(s). The chemopreventivecomposition of the invention may enhance the immune function of andprolong longevity and/or healthspan of a mammal. The chemopreventivecomposition may therefore be used in prophylaxis, prevention, andtreatment of disorders associated with, caused by or accompanied by adisease involving alteration of gene expression. In certain embodiments,the chemopreventive composition is used, e g., to prevent, delay theonset, prophylaxis or treatment of cancer, autoimmune disorders, heartdisease, inflammatory disorders, bone disorders, bladder functiondisorders, joint health disorders, arthritis, vision, depression,anxiety, Alzheimer's disease, Parkinson's disease, dementias, diabetes,coronary artery disease, kidney disease, and viral or bacterialinfections, etc. In certain embodiments, the chemopreventive compositionis used to enhance vibrancy or youthfulness or appearance of a mammaland/or improve energy in a mammal.

An immunomodulatory composition according to the invention generallycomprises an active agent selected from the group consisting ofresveratrol, genistein, ellagic acid, curcumin, quercetin, prodrugs,metabolites, salts of any of the foregoing, and mixtures of any of theforegoing (“active agents”). In certain embodiments, resveratrolcomprises from about 1% to about 80% of the immunomodulatory compositionby weight, ellagic acid may comprise from about 0.25% to about 20% ofthe immunomodulatory composition by weight, genistein comprises fromabout 0.5% to about 40% of the immunomodulatory composition by weight,curcumin comprises from about 1% to about 80% of the immunomodulatorycomposition by weight; and quercetin comprises from about 1% to about80% of the immunomodulatory composition by weight. The immunomodulatorycomposition may further comprise one or more additional agents selectedfrom the group consisting of lycopene, α-tocopherol, L-selenomethionine,ellagic acid, indole-3-carbinol, sulforaphane, allicin, daidzein, EGCG,prodrugs of any of the foregoing, metabolites of any of the foregoing,and salts of any of the foregoing. The immunomodulatory composition mayfurther comprise one or more additional antioxidant(s).

The immunomodulatory composition may inhibit H₂O₂-induced DNA strandbreakage in lymphocytes, may have a protective effect on oxidative DNAdamage, and may prevent and/or delay oxidative DNA damage. Theimmunomodulatory composition may also enhance the immune function of andprolong longevity and/or healthspan of a mammal. The immunomodulatorycomposition may therefore decrease the chances, delay the onset, treator in prophylaxis of various disorders caused by and/or associated withthe oxidative DNA damage. The immunomodulatory composition may also beused to prevent, delay the onset, treatment or prophylaxis of a diseasethat can be ameliorated through an enhancement to immune function and adisease involving alteration of gene expression. In certain embodiments,the immunomodulatory composition is used, e g., to prevent, delay theonset, prophylaxis or treatment of cancer, autoimmune disorders, heartdisease, inflammatory disorders, bone disorders, bladder functiondisorders, joint health disorders, arthritis, vision, depression,anxiety, Alzheimer's disease, Parkinson's disease, dementias, diabetes,coronary artery disease, kidney disease, and viral or bacterialinfections, etc. In certain embodiments, the immunomodulatorycomposition is used to enhance vibrancy or youthfulness or appearance ofa mammal and/or improve energy in a mammal. In certain embodiments, theimmunomodulatory composition is used to prolong longevity and/orhealthspan of a mammal (e.g., canine).

In addition, the invention is directed in part to a dog biscuitcomprising a chemopreventive composition comprising an active agentselected from the group consisting of resveratrol, genistein, ellagicacid, curcumin, quercetin, prodrugs, metabolites, salts, and mixtures ofany of the foregoing. In certain embodiments, the dog biscuit comprises:(i) resveratrol and/or a prodrug of resveratrol and/or a metabolite ofresveratrol and/or a salt of resveratrol and/or a salt of the metaboliteof resveratrol and/or a salt of the prodrug of resveratrol; (ii)genistein and/or a prodrug of genistein and/or a metabolite of genisteinand/or a salt of genistein and/or a salt of the metabolite of genisteinand/or a salt of the prodrug of genistein; (iii) ellagic acid and/or aprodrug of ellagic acid and/or a metabolite of ellagic acid and/or asalt of the ellagic acid and/or a salt of the metabolite of the ellagicacid and/or a salt of the prodrug of ellagic acid; (iv) curcumin and/ora prodrug of curcumin and/or a metabolite of curcumin and/or a salt ofcurcumin and/or the salt of the metabolite of curcumin and/or a salt ofthe prodrug of curcumin; and (v) quercetin and/or a prodrug or quercetinand/or a metabolite of quercetin and/or a salt of quercetin and/or asalt of the metabolite of the quercetin and/or a salt of the prodrug ofquercetin. A dog biscuit may, e.g., comprise from about 0.1 mg to about2000 mg, from about 100 mg to about 2000 mg, from about 200 mg to about2000 mg, from about 250 mg to about 2000 mg, from about 300 mg to about1500 mg, from about 400 mg to about 1500 mg, or from about 500 mg toabout 1500 mg of resveratrol per one biscuit; from about 0.1 mg to about1000 mg, from about 100 mg to about 1000 mg, from about 200 mg to about1000 mg, from about 250 mg to about 1000 mg, from about 300 mg to about750 mg, from about 400 mg to about 750 mg, or from about 500 mg to about750 mg of ellagic acid per one biscuit; from about 0.1 mg to about 1000mg, from about 100 mg to about 1000 mg, from about 200 mg to about 1000mg, from about 250 mg to about 1000 mg, from about 300 mg to about 750mg, from about 400 mg to about 750 mg, or from about 500 mg to about 750mg of genistein per one biscuit; from about 0.1 mg to about 2000 mg,from about 100 mg to about 2000 mg, from about 200 mg to about 2000 mg,from about 250 mg to about 2000 mg, from about 300 mg to about 1500 mg,from about 400 mg to about 1500 mg, or from about 500 mg to about 1500mg of curcumin per one biscuit; and from about 0.1 mg to about 2000 mg,from about 100 mg to about 2000 mg, from about 200 mg to about 2000 mg,from about 250 mg to about 2000 mg, from about 300 mg to about 1500 mg,from about 400 mg to about 1500 mg, or from about 500 mg to about 1500mg of quercetin per one biscuit; or equivalent amounts of metabolites,prodrugs and salts thereof. However, if necessary to achieve a desiredeffect, a higher or lower amount may be used. In these embodiments, thedog biscuit and/or the chemopreventive composition may further compriseone or more additional agents selected from the group consisting oflycopene, α-tocopherol, L-selenomethionine, ellagic acid,indole-3-carbinol, sulforaphane, allicin, daidzein, EGCG, prodrugs ofany of the foregoing, metabolites of any of the foregoing, and salts ofany of the foregoing. The dog biscuit and/or the chemopreventivecomposition may further comprise one or more additional antioxidant(s).Resveratrol, genistein, ellagic acid, curcumin, quercetin, prodrugs,metabolites, salts, and mixtures of any of the foregoing arebioavailable from the dog biscuit and are able to cross enterocytes andenter system blood circulation after oral administration of the dogbiscuit. In certain embodiments, the dog biscuit is prepared by aprocess that does not comprise baking.

In certain embodiments, a dog biscuit comprises a therapeuticallyeffective amount of a combination comprising at least three activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts, andmixtures of any of the foregoing sufficient to increase expression ofCYBB, DUS, GSR, UCP2, and VIMP genes; and downregulate ATOX1, CCL5, EPX,MPV17, PRNP and SOD3 genes in a mammal.

In certain embodiments, a dog biscuit comprises a therapeuticallyeffective amount of a combination comprising at least three or fouractive agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing sufficient to increasemonocytes and macrophages in a mammal.

In certain embodiments, a dog biscuit comprises a therapeuticallyeffective amount of a combination comprising at least three activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts, andmixtures of any of the foregoing sufficient to increase production ofIL-10, an inflammatory cytokine that decrease the production ofpro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6, in a mammal.

In certain embodiments, a dog biscuit comprises a therapeuticallyeffective amount of a combination comprising at least three activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts, andmixtures of any of the foregoing sufficient to decrease production ofIFN-γ in a mammal.

In certain embodiments, a dog biscuit comprises a therapeuticallyeffective amount of a combination comprising at least three activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts, andmixtures of any of the foregoing sufficient to decrease production ofIL-2 in a mammal.

The invention is also directed in part to a dog biscuit comprising animmunomodulatory composition comprising an active agent selected fromthe group consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts, and mixtures of any of theforegoing. In certain embodiments, the dog biscuit comprises: (i)resveratrol and/or a prodrug of resveratrol and/or a metabolite ofresveratrol and/or a salt of resveratrol and/or a salt of the metaboliteof resveratrol and/or a salt of the prodrug of resveratrol; (ii)genistein and/or a prodrug of genistein and/or a metabolite of genisteinand/or a salt of genistein and/or a salt of the metabolite of genisteinand/or a salt of the prodrug of genistein; (iii) ellagic acid and/or aprodrug of ellagic acid and/or a metabolite of ellagic acid and/or asalt of the ellagic acid and/or a salt of the metabolite of the ellagicacid and/or a salt of the prodrug of ellagic acid; (iv) curcumin and/ora prodrug of curcumin and/or a metabolite of curcumin and/or a salt ofcurcumin and/or the salt of the metabolite of curcumin and/or a salt ofthe prodrug of curcumin; and (v) quercetin and/or a prodrug or quercetinand/or a metabolite of quercetin and/or a salt of quercetin and/or asalt of the metabolite of the quercetin and/or a salt of the prodrug ofquercetin. A dog biscuit may, e.g., comprise from about 0.1 mg to about2000 mg, from about 100 mg to about 2000 mg, from about 200 mg to about2000 mg, from about 250 mg to about 2000 mg, from about 300 mg to about1500 mg, from about 400 mg to about 1500 mg, or from about 500 mg toabout 1500 mg of resveratrol per one biscuit; from about 0.1 mg to about1000 mg, from about 100 mg to about 1000 mg, from about 200 mg to about1000 mg, from about 250 mg to about 1000 mg, from about 300 mg to about750 mg, from about 400 mg to about 750 mg, or from about 500 mg to about750 mg of ellagic acid per one biscuit; from about 0.1 mg to about 1000mg, from about 100 mg to about 1000 mg, from about 200 mg to about 1000mg, from about 250 mg to about 1000 mg, from about 300 mg to about 750mg, from about 400 mg to about 750 mg, or from about 500 mg to about 750mg of genistein per one biscuit; from about 0.1 mg to about 2000 mg,from about 100 mg to about 2000 mg, from about 200 mg to about 2000 mg,from about 250 mg to about 2000 mg, from about 300 mg to about 1500 mg,from about 400 mg to about 1500 mg, or from about 500 mg to about 1500mg of curcumin per one biscuit; and from about 0.1 mg to about 2000 mg,from about 100 mg to about 2000 mg, from about 200 mg to about 2000 mg,from about 250 mg to about 2000 mg, from about 300 mg to about 1500 mg,from about 400 mg to about 1500 mg, or from about 500 mg to about 1500mg of quercetin per one biscuit, or equivalent amounts of metabolites,prodrugs and salts thereof; or equivalent amounts of metabolites,prodrugs and salts thereof. However, if necessary to achieve an adequatelevel of oxidative DNA damage protection, a higher or lower amount maybe used. In these embodiments, the dog biscuit and/or theimmunomodulatory composition may further comprise one or more additionalagents selected from the group consisting of lycopene, α-tocopherol,L-selenomethionine, ellagic acid, indole-3-carbinol, sulforaphane,allicin, daidzein, EGCG, prodrugs of any of the foregoing, metabolitesof any of the foregoing, and salts of any of the foregoing. The dogbiscuit and/or the immunomodulatory composition may further comprise oneor more additional antioxidant(s).

The invention is further directed to a dietary or nutritional supplementcomprising a chemopreventive composition comprising an active agentselected from the group consisting of resveratrol, genistein, ellagicacid, curcumin, quercetin, prodrugs, metabolites, salts, and mixtures ofany of the foregoing. In certain embodiments, the supplement comprises:(i) resveratrol and/or a prodrug of resveratrol and/or a metabolite ofresveratrol and/or a salt of resveratrol and/or a salt of the metaboliteof resveratrol and/or a salt of the prodrug of resveratrol; (ii)genistein and/or a prodrug of genistein and/or a metabolite of genisteinand/or a salt of genistein and/or a salt of the metabolite of genisteinand/or a salt of the prodrug of genistein; (iii) ellagic acid and/or aprodrug of ellagic acid and/or a metabolite of ellagic acid and/or asalt of the ellagic acid and/or a salt of the metabolite of the ellagicacid and/or a salt of the prodrug of ellagic acid; (iv) curcumin and/ora prodrug of curcumin and/or a metabolite of curcumin and/or a salt ofcurcumin and/or the salt of the metabolite of curcumin and/or a salt ofthe prodrug of curcumin; and (v) quercetin and/or a prodrug or quercetinand/or a metabolite of quercetin and/or a salt of quercetin and/or asalt of the metabolite of the quercetin and/or a salt of the prodrug ofquercetin and is suitable for administration to humans.

The invention is also directed in part to a dietary or nutritionalsupplement comprising an immunomodulatory composition comprising anactive agent selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing. In certain embodiments, thesupplement comprises: (i) resveratrol and/or a prodrug of resveratroland/or a metabolite of resveratrol and/or a salt of resveratrol and/or asalt of the metabolite of resveratrol and/or a salt of the prodrug ofresveratrol; (ii) genistein and/or a prodrug of genistein and/or ametabolite of genistein and/or a salt of genistein and/or a salt of themetabolite of genistein and/or a salt of the prodrug of genistein; (iii)ellagic acid and/or a prodrug of ellagic acid and/or a metabolite ofellagic acid and/or a salt of the ellagic acid and/or a salt of themetabolite of the ellagic acid and/or a salt of the prodrug of ellagicacid; (iv) curcumin and/or a prodrug of curcumin and/or a metabolite ofcurcumin and/or a salt of curcumin and/or the salt of the metabolite ofcurcumin and/or a salt of the prodrug of curcumin; and (v) quercetinand/or a prodrug or quercetin and/or a metabolite of quercetin and/or asalt of quercetin and/or a salt of the metabolite of the quercetinand/or a salt of the prodrug of quercetin and is suitable foradministration to humans.

The invention is further directed in part to providing a protectiveeffect on oxidative DNA damage and/or delay oxidative DNA damage inmammals (e.g., canines) by administering orally a therapeuticallyeffective amount of the immunomodulatory composition comprising anactive agent selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts of any of the foregoing, and mixtures of any of the foregoing to amammal (e.g., a canine) on a once-a-day or several times a day basis(e.g., twice-a-day or as several treats during the day). After an oraladministration of the immunomodulatory composition to a mammal (e.g.,canine), the immunomodulatory composition preferably provides detectableserum levels of, e.g., resveratrol and/or genistein metabolites and/orquercetin and/or metabolites in the mammal. Generally, resveratrol maybe administered at a daily dose of from about 0.1 grams/mammal/day toabout 2 grams/mammal/day, from about 0.2 grams/mammal/day to about 2grams/mammal/day, from about 0.3 grams/mammal/day to about 2grams/mammal/day; from about 0.4 grams/mammal/day to about 2grams/mammal/day, from about 0.5 grams/mammal/day to about 2grams/mammal/day, or from about 0.5 grams/mammal/day to about 2grams/mammal/day; genistein may be administered at a daily dose of fromabout 0.1 grams/mammal/day to 0.5 grams/mammal/day, from about 0.2grams/mammal/day to about 0.5 grams/mammal/day, or from about 0.3grams/mammal/day to about 0.5 grams/mammal/day; ellagic acid may beadministered at a daily dose of from 0.1 to 1 grams/mammal/day, fromabout 0.2 grams/mammal/day to about 1 grams/mammal/day, from about 0.3grams/mammal/day to about 1 grams/mammal/day; from about 0.4grams/mammal/day to about 1 grams/mammal/day, from about 0.5grams/mammal/day to about 1 grams/mammal/day, or from about 0.5grams/mammal/day to about 1 grams/mammal/day; curcumin may beadministered at a daily dose of from 0.1 grams/mammal/day to 2grams/mammal/day, from about 0.2 grams/mammal/day to about 2grams/mammal/day, from about 0.3 grams/mammal/day to about 2grams/mammal/day; from about 0.4 grams/mammal/day to about 2grams/mammal/day, from about 0.5 grams/mammal/day to about 2grams/mammal/day, or from about 0.5 grams/mammal/day to about 2grams/mammal/day; and quercetin may administered at a daily dose of from0.1 grams/mammal/day to 2 grams/mammal/day, from about 0.2grams/mammal/day to about 2 grams/mammal/day, from about 0.3grams/mammal/day to about 2 grams/mammal/day; from about 0.4grams/mammal/day to about 2 grams/mammal/day, from about 0.5grams/mammal/day to about 2 grams/mammal/day, or from about 0.5grams/mammal/day to about 2 grams/mammal/day. However, if necessary toachieve an adequate level of oxidative DNA damage protection, a higheror lower amount may be used.

In addition, the invention is directed in part to a method ofpreventing, treating and/or delaying an onset of a disease that can beameliorated through an enhancement to immune function comprisingadministering to a mammal in need thereof (e.g., canine) atherapeutically effective amount of the chemopreventive compositioncomprising resveratrol, genistein, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts of any of the foregoing, and mixtures ofany of the foregoing.

The invention is further directed in part to a method of preventing,treating and/or delaying an onset of a disease that can be amelioratedthrough an enhancement to immune function comprising administering to amammal in need thereof (e.g., canine) a therapeutically effective amountof the immunomodulatory composition comprising resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts of anyof the foregoing, and mixtures of any of the foregoing.

The invention if further directed in part to a method of preventing,treating, prophylaxis, and/or delaying an onset of a disease involvingalteration of gene expression in a mammal (e.g., a canine) comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of the immunomodulatory composition comprising an active agentcomprising resveratrol, genistein, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts of any of the foregoing, and mixtures ofany of the foregoing.

The invention if further directed in part to a method of preventing,treating, prophylaxis, and/or delaying an onset of a disease involvingalteration of gene expression in a mammal (e.g., a canine) comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of the chemopreventive composition comprising an active agentcomprising resveratrol, genistein, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts of any of the foregoing, and mixtures ofany of the foregoing

The invention is further directed in part to a method of enhancingvibrancy, youthfulness or appearance of a mammal (e.g., a canine)comprising administering to a mammal in need thereof a therapeuticallyeffective amount of the immunomodulatory composition comprising anactive agent consisting of resveratrol, genistein, ellagic acid,curcumin, quercetin, prodrugs, metabolites, salts of any of theforegoing, and mixtures of any of the foregoing.

The invention is also directed in part to a method of enhancingvibrancy, youthfulness or appearance of a mammal (e.g., a canine)comprising administering to a mammal in need thereof a therapeuticallyeffective amount of the chemopreventive composition comprising an activeagent consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing.

In addition, the invention is directed in part to a method of improvingenergy in a mammal (e.g., a canine) comprising administering to a mammalin need thereof a therapeutically effective amount of theimmunomodulatory composition comprising an active agent selected fromthe group consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing.

The invention is directed in part to a method of improving energy in amammal (e.g., a canine) comprising administering to a mammal in needthereof a therapeutically effective amount of the chemopreventivecomposition comprising an active agent selected from the groupconsisting of resveratrol, genistein, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts of any of the foregoing, and mixtures ofany of the foregoing.

The invention is also directed to a method of prolonging longevity of amammal to a mammal in need thereof a therapeutically effective amount ofa composition comprising at least three active agents selected from thegroup consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing.

The invention is also directed to a method of prolonging healthspan of amammal to a mammal in need thereof a therapeutically effective amount ofa composition comprising at least three active agents selected from thegroup consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing.

In the methods of the present invention, the antioxidative stresscomposition (e.g., the chemopreventive or the immunomodulatingcomposition) may be administered orally as part of a conventional solidpharmaceutical dosage form (e.g., as a tablet, capsule, or powder), as aliquid (a solution, an emulsion or a suspension), or as a food additive,a dog chow, or as a dog biscuit or a biscuit-type dosage form. Theantioxidative stress composition may be administered once-a-day,twice-a-day, three times a day, four times a day, or as needed (e.g., aspart of a treat). In certain embodiments, the composition isadministered with an additional active agent. In certain embodiments,the administration would start at the time of full growth of a mammal.For canine, this would be roughly 1 year for smaller breeds, and 2 yearsfor larger breeds. Once started, the administration could continue for aperiod of from about 3 months to about 20 years.

The invention is further directed in part to a method of preparing animmunomodulatory composition comprising an active agent selected fromthe group consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing. In certain embodiments at least threeactive agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts of any of the foregoing, and mixtures of any of the foregoing areincorporated into the immunomodulatory composition.

The invention is also directed in part to a method of preparing achemopreventive composition comprising an active agent selected from thegroup consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing. In certain embodiments at least threeactive agents selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts of any of the foregoing, and mixtures of any of the foregoing areincorporated into the chemopreventive composition.

The invention is further directed in part to a method of preparing a dogbiscuit comprising an immunomodulatory composition comprising an activeagent selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts of anyof the foregoing, and mixtures of any of the foregoing. In certainembodiments at least three active agents selected from the groupconsisting of resveratrol, genistein, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts of any of the foregoing, and mixtures ofany of the foregoing are incorporated into the dog biscuit. Generally, amix which is free from the immunomodulatory composition is prepared andbaked, and then the immunomodulatory composition or the components ofthe immunomodulatory composition are incorporated into the mix after themix has been already baked. In certain embodiments, the methods avoidthe conventional practice of baking the final product.

In addition, the invention is directed in part to a method of preparinga dog biscuit comprising a chemopreventive composition comprising anactive agent selected from the group consisting of resveratrol,genistein, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts of any of the foregoing, and mixtures of any of the foregoing.Generally, a mix which is free from the chemopreventive composition isprepared and baked, and then the chemopreventive composition or thecomponents of the chemopreventive composition are incorporated into themix after the mix has been already baked. In certain embodiments, themethods avoid the conventional practice of baking the final product.

It is believed that the combinations of active agents described hereinand used in the chemopreventive and immunomodulatory compositions of theinvention have not been used and tested prior to the present invention.The combinations of the active agents used in the chemopreventive andimmunomodulatory compositions of the invention are therefore new andunique.

As described herein, a number of factors were taken into account inmaking the selections of the active agents of the chemopreventive andimmunomodulatory compositions of the invention. The results reportedherein show significant promise and support the notion of providing andperfecting chemopreventive compositions, immunomodulatory compositionsand nontoxic dietary supplements can be of value in enhancing the healthand well-being of mammals (e.g., canines) and/or improving longevity ofmammals (e.g., canines). Of course this would be a meaningfulaccomplishment for the dog him- or herself, but owners of domestic petsand service animals would also derive great benefit. Further, the workwith canines may provide some insight relevant to and may beextrapolated to human disease prophylaxis, prevention and treatment.

Definitions

The term “about” in the present specification means a value within 15%(±15%) of the value recited immediately after the term “about,”including the value equal to the upper limit (i.e., +15%) and the valueequal to the lower limit (i.e., −15%) of this range. For example, thephrase “about 100” encompasses any numeric value that is between 85 and115, including 85 and 115.

The term “healthspan” means the length of time in one's life where oneis in optimal health. The healthspan may, e.g., be from 1 year to 90years. For cannines, the healthspan may, e.g., be from 1 year to 25years.

The term “mean” as used herein is calculated from the data of eightsubjects (e.g., canines).

The term “oral administration” encompasses administration with food andwithout food.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that compositions according to the invention inhibitH₂O₂-induced DNA strand breakage in dog lymphocytes. Lymphocytes werewashed, suspended in 1% (w/v) LMP agarose and pipetted onto frostedglass microscope slides. For H₂O₂ treatment, 10 μl of 1 mM H₂O₂ wereadded to 90 μl of cells in PBS (100 μM final concentration); after 5 minon ice, the cells were collected by centrifugation and applied toslides. Slides were washed three-times with buffer (40 mM HEPES-KOH, 0.1M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8) and incubated for 45 min at 37°C. with either 50 μl buffer or endonuclease III in buffer (1 μgprotein/ml). Lymphocytes were stained with SYBR Gold and image analysiswas performed using a Leica confocal microscope (FIG. 2). Gray bars arewithout endonuclease treatment, black bars are with treatment. **p<0.01,t-test (n=16).

FIG. 2 shows heat map of oxidative stress genes expression in doglymphocytes. Lymphocytes were isolated from the same dogs after the twophases of the study. RNA was extracted and reverse SyBR Green detectionsystem. The thermal cycling condition was 95° C. for 10 min and then 40cycles at transcribed to cDNA. Then, cDNA was mixed with RT2 qPCR MasterMix and 25 μl were placed in each well of the array plate. Real-timequantitative PCR was performed using the 95° C. for 15 s followed by 60°C. for 1 min. The fold changes between the two phases of the study wereanalyzed using the Web-based software provided by SABiosciences (RT2Profiler PCR Array Data Analysis). Fold changes of 84 genes weredetermined and presented as heatmaps (Red: up-regulation, green:down-regulation).

FIG. 3 shows comet images of lymphocyte DNA damage from control dogs anddogs receiving dietary supplementation. Frozen lymphocytes were thawed,centrifuged, resuspended in PBS and incubated 5 min on ice with 100 μMH₂O₂. DNA damage was measured with the comet assay. Type 0, no DNAdamage detected in lymphocytes; type 1 and 2 presented at the most inlymphocytes with endogenous DNA damage; type 3 and 4 are in H₂O₂ orendonuclease III inducible DNA damage.

DETAILED DESCRIPTION

Oxidative stress causes damage to DNA, RNA, proteins, and other cellularcomponents. Oxidative stress is therefore thought to be a source of manydisorders, including, e.g., aging, cancer, autoimmune disorders, heartdisease, inflammatory disorders, bone disorders, bladder functiondisorders, joint health disorders, arthritis, vision, depression,anxiety, Alzheimer's disease, Parkinson's disease, dementias, diabetes,coronary artery disease, kidney disease, and viral or bacterialinfections.

Oxidative DNA damage may also adversely affects mammals and may causecancers and tumors.

Similar to humans, dogs manifest a broad range of cancers such asmelanoma, non-Hodgkin lymphoma, osteosarcoma, soft tissue sarcoma, andprostate, mammary, lung and colorectal carcinomas. Approximately 1 in 3dogs will be diagnosed with cancer during their lifetime, and cancercurrently accounts for about half of the deaths of all dogs older than10 years^(2,36).

In situations wherein a tumor is diagnosed and completely excised priorto invasion or metastatic spread, treatment should be definitive. Incanine cases, this may involve amputation or radical surgery. However,the situation is even worse when presented with malignant metastaticdisease wherein life expectancy is reduced and treatment options aremuch more limited. Consequently, avoiding all such situations,ostensibly by means of cancer chemoprevention, remains a compellingapproach. Despite issues associated with the development of cancerchemopreventive agents, proof-of-principal has been established withdrugs such as tamoxifen and finasteride³⁵. In nearly every case, for thediscovery or development of new cancer chemopreventive agents,laboratory animals (primarily rodents) have been used as models. In moreadvanced stages of development, additional animals, including canines,are used to establish pharmacokinetics (absorption, distribution,metabolism, and excretion), dosage forms, toxicity, etc. Nonetheless,the ostensible goal of such work is to provide agents for the preventionof cancer with human beings.

In contrast, in the study described below, rather than using the canineas a model for human beings, a goal was also to focus on potentialbenefits for the animal.

Chemoprevention is an attempt to use nontoxic natural and syntheticsubstances or their mixtures (e.g., immunomodulatory compositions) tointervene during the relatively early stages of carcinogenesis, beforeinvasive characteristics are manifested⁵. Since carcinogenesis isregarded as a multistep process (e.g., initiation, promotion,progression)⁶, in principle, blocking or inhibiting any of these stagescould help to prevent or delay tumorigenesis. Further, consistent withepidemiological studies suggesting that a reduced risk of cancer isassociated with consumption of vegetables and fruits⁷⁻¹¹, many agentswith cancer chemopreventive potential are naturally occurringphytochemicals¹²⁻¹⁴. The etiologic basis of cancer, as well as mostage-related diseases, is complex, but it is generally agreed thatoxidative stress plays a role¹⁵. Damage to DNA by oxidative stress iswell known¹⁶. An aim of the study described below was to assess whetherdietary intake of some common dietary chemopreventive agents couldaffect DNA damage and the expression of genes related to oxidativestress with dog lymphocytes.

As described in detail in the Examples section below, detectable serumlevels of resveratrol, genistein metabolites, and quercetin (andmetabolites) were demonstrated after administration of theimmunomodulatory compositions described herein, indicating they crosscanine enterocytes and enter blood circulation. This promoted aprotective effect on oxidative DNA damage. A hypothesis is thatlymphocytes serve as a surrogate for other cell types, and protectionfrom DNA damage is consistent with cancer chemoprevention. The mechanismof protection remains to be defined, but to provide some indication ofthe effect of the chemopreventive formulation on oxidative stress geneexpression, array analysis was performed. Of the up-regulated genes,three belong to oxidative stress responsive group (DUSP1, GSR, VIMP),CYBB is an antioxidant, and UCP2 is related to superoxide metabolism.Many selenoproteins (VIMP) participate in intracellular redoxhomeostasis and play antioxidant roles⁴⁹. The expression ofselenoprotein S (SelS) is related to inflammation and insulin resistancesuggesting that SelS may provide a link between inflammation andoxidative stress pathways through its role as an antioxidant⁵⁰.Down-regulated genes are represented by two genes (ATOX1 and MPV17)associated with ROS metabolism, two oxidative stress responsive genes(CCL5 and PRNP), and two others from the antioxidant group (SOD3 andEPX)^(36,51-54) (Table 6). Overall, in the context of gene expression,up-regulation (e.g., GSR and UCP2) and down-regulation (e.g., SOD3 andthe transcription factor for SOD3, Atox1)⁵⁵ may improve antioxidantresponse or decrease inflammation.

Although, DNA was used as a biomarker in the experiments below, it isbelieved that the compositions of the invention would provide similaranti-oxidative stress effects in other cellular components, organs andtissues. For example, it is believed that a similar anti-oxidativestress effect is exhibed by the compositions of the invention in, e.g.,liver and brain cells.

Of course from a humanitarian point-of-view prevention of canine canceris gratifying, but owners of pets and service animals would alsobenefit.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following examples illustrate various aspects of the presentinvention. They are not to be construed to limit the claims in anymanner whatsoever. Additional components can be added to theantioxidative stress compositions of the invention, including thechemoprotective compositions and immunomodulatory compositions, based onthe judgement of those ordinary experienced in the art.

Example 1

This Example documents the first report to document oral administrationof this schedule of combined chemopreventive agents in a canine study.

In enrolling canine pets as the subjects of the study, an objective wasto perform a relatively short-term and non-invasive trial. Accordingly,lymphocytic DNA was used as a surrogate biomarker for oxidative stress.As described in the literature, this procedure has been used forassessing both the carcinogenic and anti-carcinogenic potential¹⁷ oftest substances and, although lymphocytes are used for the observation,it may be surmised that other cell-types susceptible to carcinogenicinsult could mount a similar response¹⁷. Since the ex vivo protectiveresponse of the lymphocyte is evaluated, and the chemopreventive agentis administered orally, it is logical to assume any positive responseelicited by the agent would be through an indirect mechanism such asinduction of cytoprotective enzymes. Thus, expression profiles with anarray designed for canine oxidative stress genes were determined.

During the course of the study, however, an increase in blood monocytecount was observed. This increase was not outside the bounds of what isconsidered in the normal range by those in the practice of veterinarymedicine but, nonetheless, the increase was statistically significant.Bearing in mind the ever increasing role of immunomodulation in cancerprevention or treatment¹⁸, it was decided to perform limited tests toquantify plasma levels of select cytokines. The levels of INF-γ, IL-2,and IL-10 were measured. Members of the interleukin family are involvedin cellular defense against viral pathogens playing an important role inresponses resulting from oxidative stress. The multifaceted nature ofIFN-γ points toward numerous possible therapeutic targets that have thepotential of producing significant benefits¹⁹. Indeed, almost 70% ofgenes regulated by IFN-γ are also regulated by type I IFN. Of theremaining nonredundant genes affected by IFN-γ, over two-thirds areinducible and the remainder is repressed. Thus, IFN-γ uniquely regulatesa significant number of genes related to inflammation and oxidativestress²⁰. For many years, the role of IL-2 was established as a cytokinepossessing strong pro-inflammatory activity. More recently, however,studies have suggested a role for IL-2 in autoimmune chronicinflammation, suggesting a protective function, when administered atlow-doses²¹. IL-10 is an anti-inflammatory cytokine that decreases theproduction of pro-inflammatory cytokines, such as TNF-α, IL-1β, andIL-6, produced by macrophages or monocytes²².

In order to conduct the canine trial, a biscuit containing five agents:resveratrol, ellagic acid, curcumin, genistein and quercetin wasformulated and manufactured. When administered as single agents, somestudies with dogs have been reported in the literature. However, thein-vivo performance of a composition comprising resveratrol, ellagicacid, curcumin, genistein and quercetin is not predictable form theliterature. It is also not predictable from the literature howresveratrol, ellagic acid, curcumin, genistein and quercetin will behavewhen used together in a combination.

For example, plasma levels of genistein were determined in Beagle dogstreated with immediate and extended release tablets²³, dogs were treatedwith curcumin for the potential therapeutic management ofosteoarthritis²⁴, and quercetin, in combination with clopidogrel,affected the activity of P-glycoprotein²⁵. Of course, many preclinicalstudies have been performed with dogs to assess potential toxicity as aprelude to conducting human trials, such as with resveratrol²⁶⁻²⁸.

The chemopreventive formulation devised for this study was based on avariety of considerations. First of all, it was of interest to selectagents that (1) are natural products, (2) ostensibly function bypleotropic mechanisms (e.g., inhibit various stages of carcinogenesissuch as initiation, promotion, and progression, either singly or incombination), (3) have well-known safety profiles or generallyrecognized as safe (GRAS) status, (4) have been extensively reported inthe scientific literature as chemopreventive agents, and (5) areundergoing or have undergone evaluation in human clinical trials. Afterthorough evaluation, the following agents were judged as having metthese criteria: Resveratrol (‘typical’ representative dose, 1g/d/person), lycopene (15 mg/d), α-tocopherol (50 mg/d),L-selenomethionine (0.2 mg/d), ellagic acid (250 mg/d),indole-3-carbinol (400 mg/d), sulforophane (50 mg/d), quercetin (1 g/d),allicin (3 mg/d), genistein (500 mg/d), daidzein (300 mg/d), curcumin (1g/d), and EGCG (300 mg/d). At this point in the selection process, someagents were eliminated since adequate quantities may be found in theaverage diet (e.g., vitamin D, selenium, and α-tocopherol), high expenseor issues with general availability (e.g., sulforaphane), poororganoleptic properties (e.g., allicin), or innate difficulty in thebiscuit production process.

In the end, a combination including resveratrol, genistein, ellagicacid, curcumin and quercetin was selected for administration at thedosage levels summarized in Table 1:

TABLE 1 Chemopreventive compounds administered to dogs. Dose in Dosehuman (g/dog/ studies Compound Source Purity day) (g/person/day)Resveratrol Hangzhon >95% 0.5 1.0 Dayangchem Co., Shanghai, ChinaEllagic Shandong Juye >99% 0.125 0.25 acid Sunnyfarm Natural ProductCO., LTD, Shandong, China Genistein Shandong Juye >95% 0.250 0.5Sunnyfarm Natural Product CO., LTD, Shandong, China Curcumin ShandongJuye >95% 0.5 1.0 Sunnyfarm Natural Product CO., LTD, Shandong, ChinaQuercetin Hangzhon >95% 0.5 1.0 Dayangchem Co., Shanghai, China

The rationale of the dosage selection is described in Methods (Dogbiscuit production). Additional or alternative agents could also havebeen included in this trial. Nonetheless, in addition to therationalization presented above, another advantage was that each of thetest agents is known to function by pleiotropic mechanisms of actionthat are relevant to disease prevention. For example, resveratrol hasbeen reported to exert antioxidant, anti-inflammatory, anti-infective,cardioprotective, neuroprotective, antiobesity, and chemopreventiveactivities³⁷⁻⁴⁰. It has not been evaluated for cancer chemoprevention indogs, but analytical work for the determination of trans-resveratrol andderivatives in dog plasma has been described^(26,27), as well aspreclinical toxicity studies²⁸. Ellagic acid occurs in many foods andhas antioxidant, antibacterial, antiviral and cancer-preventingproperties⁴¹. It exerts anticancer effects in various cancer cell linesand animal tumor models⁴². Curcumin, a natural polyphenolic antioxidantcan mediate a large number of biological responses⁴³. Some studies havereported pharmacokinetics in dogs, and stabilizing curcumin withphosphoric acid allows accurate quantitative determinations ofcurcuminoids in dog plasma⁴⁴. The phytoestrogen genistein is found insoy-based products. It has been suggested that genistein can preventboth prostate and breast cancer³³, particularly in Southeast Asiapopulations where soy products are consumed at high levels⁴⁵. A LC-MS-MSmethod has been developed for the determination of genistein in dogplasma following oral administration²³. Finally, quercetin mediatesdiverse anti-cancer effects⁴⁶. It is a pleiotropic molecule with limitedtoxicity on normal cells. Simultaneous targeting of multiple pathwaysmay help to eliminate malignant cells and retard the onset of drugresistance⁴⁷.

Example 2

The development and production of a biscuit-type dosage form forrelatively large canines presented some challenges since it wasimportant to preserve the integrity of the chemopreventive agents. Thus,the common practice of baking the final product was avoided. During thedevelopment phase, many formulations were designed to form durablebiscuit-type tablets and changes were made in an effort to reducebellyband cracks and improve compressibility, compatibility, andflavour. The compaction of each active ingredient was individuallytested, which revealed that the compounds themselves aid the compressionof the tablets and helped balance the unfavourable compactioncharacteristics of the other biscuit ingredients.

Another issue that will ultimately be faced in developing a caninechemopreventive product is expense. Based on the quantities and priceschedule for the chemopreventive agents purchased and used in thisstudy, the expenditure was $1.65/biscuit. It is difficult to know anamount consumers would find to be acceptable for such as product, butgiven the high expense associated with treatment (vide supra) and thegratification accompanying disease prevention, an expenditure of thismagnitude seems reasonable. Another aspect related to expense is whenthe dog would begin biscuit consumption. While it is not possible toprovide an answer based on existing evidence, since it has been notedthat more than half of dogs over 10 years of age are likely to developcancer⁴⁸, it would seem that early administration would be beneficial.One suggestion would be to start consumption at the time of full growth,which would be roughly 1 year for smaller breeds, and 2 years for largerbreeds.

Nevertheless, a dog biscuit formulation was devised that contained acombination of resveratrol, genistein, ellagic acid, curcumin andquercetin as active chemopreventive agents. The study design involvedtwo phases. The first phase was to evaluate the palatability andacceptability of the placebo treats. Over a period of three weeks, eighthealthy dogs were given the placebo treat twice per day. This assessmentwas simply to determine whether the dog liked/disliked the taste,texture, etc., of the biscuit, the owner's ease of administration, andthe dog's motivation to eat the biscuit. The owners observed thebehaviour of the dog to insure the biscuits were well tolerated with andwithout active ingredients to ensure their delivery. The dogs consumedthe biscuits following the prescribed schedule. Some owners used maskingtechniques.

Blood samples were drawn at the end of the first phase of the study.Following a two-month resting period, the second phase of the study waslaunched with the same canine subjects. Over a period of three weeks,the diets of the dogs were supplemented with two biscuit treatscontaining the chemopreventive agents (Table 1), one biscuit by mouthtwice daily approximately 12 hours apart each day. After anadministration period of three weeks, blood was drawn, and the study wasterminated. The comprehensive blood profiles are depicted in Table 2.

TABLE 2 Comprehensive blood profiles. Normal First Second Range phasephase ALB 2.5-4.4 g/dL 3.6 ± 0.1 3.6 ± 0.1 ALP 20-150 U/L 45.1 ± 6.9 45.5 ± 6.1  ALT 10-118 U/L 53.1 ± 9.4  69.1 ± 11.5 AMY 200-1200 U/L511.1 ± 53.6  538.6 ± 58.7  TBIL 0.1-0.6 mg/dL 0.3 ± 1.0 0.3 ± 0  BUN7-25 mg/dL 14.3 ± 1.0  15.4 ± 1.2  CA++ 8.6-11.8 mg/dL 10.6 ± 0.1  10.6± 0.1  PHOS 2.9-6.6 mg/dL  3.7 ± 0.2*  4.6 ± 0.1* CRE 0.3-1.4 mg/dL 1.1± 0.1 1.0 ± 0.1 GLU 60-110 mg/dL 104.6 ± 4.0  104.4 ± 4.5  NA+ 138-160mmol/L 144.0 ± 0.7  145.3 ± 0.3  K+ 3.7-5.8 mmol/L 4.7 ± 0.2 4.2 ± 0.1TP 5.4-8.2 g/dL 6.7 ± 0.1 6.7 ± 0.1 GLOB 2.3-5.2 g/dL 3.1 ± 0.1 3.3 ±0.1 ALB—albumin; ALP—alkaline phosphatase; ALT—alanine aminotransferase;AMY—amylase; TBIL—total bilirubin; BUN—blood urea nitrogen;CA++—calcium; PHOS—phosphorus; CRE—creatinine; GLU—glucose; NA+—sodium;K+—potassium; TP—total protein; GLOB—globulin. *p < 0.01.

The owners of these pets transported the dogs to a local veterinaryclinic on pre-scheduled days. During these visits, the dogs were weighedand received physical examinations. Venous blood was used forcomprehensive serum chemistry profiles and complete blood counts toevaluate organ function and health status, as well as analysis of thechemopreventive agents and assessment of biomarkers. The dogs weremaintained by their owners and not housed in any research facility.Special instructions were given as a caution label for biscuits such askeep away from children, store in a refrigerator, may turn urine, feces,saliva yellow, give only as directed, and for animal consumption only.Dogs did not experience any overt toxicity while enrolled in trial.There were no reported changes in bowel habits (diarrhea orconstipation), no abdominal bloating, and no gastrointestinal bleeding.However, some minor gastrointestinal effects were reported by theowners. Of the eight canine subjects enrolled in the study, one ownerreported decreased appetite while on the biscuit, one owner reportedmustard-colored stools while on the biscuit, and three owners reportedvomiting following administration of the biscuit that was sporadic.

Haematological parameters were assessed and all values were observed tofall within the normal range attesting to a general state of good healthboth before and after dietary supplementation.

Comprehensive blood profiles did not show any difference between thefirst and second phases of the study falling outside of the normal range(Table 2). However, there was a significant increase in the level ofserum phosphorous. The reason for this increase is not known. However, alow magnitude change in a single marker wherein the total level stillremains in the normal range is not of concern from a veterinary medicinepoint-of-view. In sum, these data suggest the treatment protocol did notlead to any type of overt toxicity.

The same was true when comparing baseline and post-treatment bloodcounts (Table 3).

TABLE 3 Complete blood count. Normal First Second Range phase phase GRA3.0-12.0 × 10⁹/L   8.4 ± 0.71  9.9 ± 0.8 WBC 6.0-17.0 × 10⁹/L  11.1 ±1.1 12.7 ± 1.2 LYM 1.0-4.8 × 10⁹/L  2.2 ± 0.6  1.9 ± 0.5 MON 0.2-1.5 ×10⁹/L  0.5 ± 0.1*  0.9 ± 0.1* RBC  5.5-8.5 × 10¹²/L  7.8 ± 0.3  7.4 ±0.3 HGB 12.0-18.0 g/dL 16.9 ± 0.6 17.0 ± 0.4 HCT 37.0-55.0% 49.2 ± 1.949.3 ± 2.1 MCV 60-77 fl 65.6 ± 1.2 66.3 ± 1.1 MCH 19.5-24.5 pg 23.2 ±1.0 23.0 ± 0.4 MCHC 31.0-34.0 g/dL 34.3 ± 0.5 34.7 ± 0.7 PLT 200-500 ×10⁹/L  233.8 ± 25.5 272.6 ± 59.4 MPV 3.9-11.1 fl 10.1 ± 0.4  9.9 ± 0.4GRA—granulocytes; WBC—white blood cells; LYM—lymphocytes; MON—monocytes;RBC—red blood cells; HGB—hemoglobin; HCT—hematocrit; MCV—meancorpuscular volume; MCH—mean corpuscular hemoglobin; MCHC—meancorpuscular hemoglobin concentration; PLT—platelets; MPV—mean plateletvolume; *p < 0.0001

It is notable, however, when comparing these two groups, there was astatistically significant increase in the number of monocytes after thedogs were given the dietary supplement for the treatment period.Although remaining in the normal range, the monocyte number was found tobe significantly higher in the second phase of the study (vide infra)(Table 3).

Serum was isolated from whole blood obtained during the two phases ofthe trial and analysed by LC-MS/MS. The concentrations ofchemopreventive compounds and their metabolites is depicted in FIG. 4:

TABLE 4 Concentrations of chemopreventive compounds and its metabolitesin dog serum. Concentration in Concentration in serum, ng/ml serum,ng/ml Compounds First phase Second phase Resveratrol N/D  19.3 ±11.02^(a) Resveratrol-3-O-glucuronide N/D  64.29 ± 27.24^(a)trans-Resveratrol-4′-sulfate N/D  44.43 ± 12.23^(a)trans-Resveratol-3-sulfate N/D 424.38 ± 80.40^(a)cis-Resveratrol-3-sulfate N/D 13.63 ± 5.27^(a) Resveratrol sulfate totalN/D 482.43 ± 75.60^(a) Ellagic acid N/D N/D Genistein N/D N/DGenistein-4′-glucuronide 327.72 ± 211.13^(a)   7083.7 ± 128.18*Genistein-7-glucuronide N/D 861.74 ± 329.6  Genistein sulfate  961 ±680.3 26958.6 ± 2300**  Curcumin N/D N/D Curcumin glucuronide N/D N/D^(b) Quercetin  N/D^(c)  2 ± 1 N/D—none detected; ^(a)one dog didnot show any resveratrol, genistein, or respective metabolites, in bloodserum, and was excluded from the analysis; ^(b)two dogs hadconcentration of curcumin glucuronide 94 and 31 ng/ml; ^(c)three dogshad concentration of quercetin 2, 3 and 2 ng/ml, all others dogs hadbelow detectable level; *p = 0.0018; **p = 0.003.

The chemopreventive agents used for this are not normally added to dogchow, and selected dosages were well within safety margins based onprevious work conducted mainly in human clinical trials. At the end ofthis first (placebo) phase of the trial, no resveratrol, resveratrolmetabolites, curcumin, curcumin metabolites, or ellagic acid weredetected in serum. Genistein-4′-glucuronide (327.7±211.13 ng/mL) andgenistein sulfate (961±680.1 ng/mL) were detected in serum of six dogs.It is likely the genistein metabolites were derived from the dog chow,which can contain soybean products. Quercetin levels were belowdetectable levels in five dogs, but three dogs had quercetinconcentrations of 2 or 3 ng/mL. Quercetin occurs in many botanicals andwas also probably a constituent of the dog chow (Table 4). After atwo-month rest (wash-out) period, the dogs were started on the secondphase of the trial by administering two biscuits per day containing thechemopreventive agents. The second phase lasted 3 weeks. Resveratrol andits metabolites were detected in serum of all dogs but one during thesecond phase. The most abundant form of resveratrol in dog serum wasresveratrol-3-sulfate. Much higher concentrations of genisteinmetabolites were observed during the second phase(genistein-4′-glucuronide, 7083.74±128.18 ng/ml;genistein-7-glucuronide, 861.74±329.61 ng/ml), with the sulfate beingdominant (genistein sulfate, 26958.61±2300 ng/ml) (Table 4). A diet richin genistein has been associated with lower rates of prostate and breastcancer³³. When administered orally, genistein is rapidly absorbed andmetabolized in humans and animals as are other isoflavones³⁴. In thecurrent study, no unconjugated genistein was detected after either thefirst or the second phases (Table 4). No ellagic acid or curcumin wasdetected in serum, although curcumin glucuronide was detected at lowlevels in serum of two dogs after the second phase.

Starting with whole blood from the canine subjects, peripheral bloodlymphocytes were isolated. Two measures of DNA damage were evaluated:endogenous DNA damage (DNA damage without H₂O₂ exposure) andH₂O₂-inducible DNA damage. No statistically significant difference inendogenous lymphocyte DNA damage between the two phases of the study wasfound (FIG. 1). In dog lymphocytes obtained during the first phase ofthe trial, H₂O₂-induced DNA strand breakage increased from 54±15 to310±30 units. Remarkably, in second phase of the trial, followingadministration of the dietary supplement, lymphocyte DNA damage inducedby treatment with 100 μM H₂O₂ was significantly decreased from 310±30 to180±16 units (FIG. 1, grey bars). For further confirmation of thesedata, cell preparations were treated with endonuclease III and themeasurements were repeated. Levels of endogenous oxidized pyrimidinesdid not differ during the first or second phases of the trial. However,H₂O₂ treatment increased the level of oxidized pyrimidines, andconsumption of the dietary supplement protected lymphocytes againstpyrimidine oxidation (FIG. 1, black bars).

To further explore the ramifications of chemopreventive agentadministration, total RNA was extracted from lymphocytes, cDNA wasgenerated, and evaluated using a custom made PCR array relevant to dogoxidative stress (Table 5). The oxidative stress related genes custommade for dogs are depicted in Table 5:

TABLE 5 Oxidative stress related genes custom made for dogs. GeneBankSymbol Description NM_001003026 ALB Albumin XM_536613 ALOX12Arachidonate 12-lipoxygenase XM_845955 AOX1 Aldehyde oxidase 1pseudogene XM_860950 APOE Apolipoprotein E NM_001003119 ATOX1 ATX1antioxidant protein 1 homolog (yeast) XM_845605 BNIP3L BCL2/adenovirusE1B 19 kDa protein 3-like NM_001002984 CAT Catalase NM_001003010 CCL5Chemokine (C-C motif) ligand 5 NM_001194970 CCS Copper chaperone forsuperoxide dismutase NM_001100290 CYBA Cytochrome b-245, alphapolypeptide NM_001100291 CYBB Cytochrome b-245, beta polypeptideNM_001077587 CYGB Cytoglobin XM_546693 DHCR24 24-dehydrocholesterolreductase NM_001003122 DUOX1 Dual oxidase 1 XM_005638367 DUOX2 Dualoxidase 2 XM_005619446 DUSP1 Dual specificity phosphatase 1 XM_005635675EPHX2 Epoxide hydrolase 2, cytoplasmic XM_548229 EPX Eosinophilperoxidase XM_005637294 FOXM1 Forkhead box M1 NM_001003080 FTH1Ferritin, heavy polypeptide 1 XM_003431750 GCLC Glutamate-cysteineligase, catalytic subunit XM_005621897 GCLM Glutamate-cysteine ligase,modifier subunit NM_001115119 GPX1 Glutathione peroxidase 1 NM_001115135GPX2 Glutathione peroxidase 2 (gastrointestinal) NM_001164454 GPX3Glutathione peroxidase 3 NM_001003213 GPX5 Glutathione peroxidase 5(epididymal androgen-protein) NM_001256320 GPX6 Glutathione peroxidase 6(olfactory) XM_005629410 GPX7 Glutathione peroxidase 7 NM_001252323 GPX8Glutathione peroxidase 8 (putative) XM_003432097 GSR Glutathionereductase XM_005634964 GSS Glutathione synthetase NM_001252167 GSTP1Glutathione S-transferase pi 1 XM_003435052 GSTZ1 Glutathionetransferase zeta 1 XM_005620945 GTF2I General transcription factor IIiNM_001194969 HMOX1 Heme oxygenase (decycling) 1 NM_001003392 KRT1Keratin 1 XM_005627164 LOC102155697 Heat shock 70 kDa protein 1-likeXM_844054 LOC607408 BCL2/adenovirus E1B 19 kDa interacting protein 3XM_003433333 LOC608103 Methionine sulfoxide reductase A XM_548231 LPOLactoperoxidase XM_005625921 MB Myoglobin NM_001252410 MGST3 Microsomalglutathione S-transferase 3 XM_847352 MPO Myeloperoxidase XM_005630257MPV17 Mp V17 mitochondrial inner membrane protein XM_844481 NCF1Neutrophil cytosolic factor 1 NM_001101832 NCF2 Neutrophil cytosolicfactor 2 NM_001003186 NOS2 Nitric oxide synthase 2, inducibleXM_005633778 NOX4 NADPH oxidase 4 NM_001103218 NOX5 NADPH oxidase,EF-hand Ca binding domain 5 XM_848524 NQO1 NAD(P)H dehydrogenase,quinone 1 XM_547012 NUDT1 Nudix (nucleoside diphosphate moiety X)-typemotif 1 XM_003431771 OXR1 Oxidation resistance 1 XM_005634251 OXSR1Oxidative-stress responsive 1 XM_534974 PDLIM1 PDZ and LIM domain 1XM_005616288 PNKP Polynucleotide kinase 3′-phosphatase NM_001252165PRDX1 Peroxiredoxin 1 XM_542042 PRDX2 Peroxiredoxin 2 NM_001256485 PRDX3Peroxiredoxin 3 XM_548896 PRDX4 Peroxiredoxin 4 XM_005631542 PRDX5Peroxiredoxin 5 XM_537190 PRDX6 Peroxiredoxin 6 XM_543041 PREX1Phosphatidylinositol-3,4,5- dependent Rac exchange factor 1 NM_001013423PRNP Prion protein NM_001003023 PTGS1 Prostaglandin-endoperoxidesynthase 1 NM_001003354 PTGS2 Prostaglandin-endoperoxide synthase 2XM_005637964 PXDNL Peroxidasin homolog (Drosophila)-like XM_543225SCARA3 Scavenger receptor class A, member 3 NM_001115118 SEPP1Selenoprotein P, plasma, 1 XM_546184 SFTPD Surfactant protein DXM_850289 SIRT2 Sirtuin 2 NM_001003035 SOD1 Superoxide dismutase 1,soluble XM_533463 SOD2 Superoxide dismutase 2, mitochondrial XM_545973SOD3 Superoxide dismutase 3, extracellular XM_005626348 SQSTM1Sequestosome 1 XM_005635344 SRXN1 Sulfiredoxin 1 NM_001286859 STK25Serine/threonine-protein kinase 25 NM_001003009 TPO Thyroid peroxidaseXM_535981 TTN Titin XM_533037 TXNIP Thioredoxin interacting proteinNM_001122673 TXNRD1 Thioredoxin reductase 1 XM_845088 TXNRD2 Thioredoxinreductase 2 NM_001122778 TXNRD3 Thioredoxin reductase 3 NM_001003048UCP2 Uncoupling protein 2 (mitochondrial, proton carrier) NM_001114757VIMP Selenoprotein S

Of the 84 genes evaluated, the expression of 11 was found to besignificantly altered following administration of the dietarysupplement. These 11 genes are listed in Table 6:

TABLE 6 Transcriptional regulation of oxidative stress-related genes indog lymphocytes. Fold P Gene Description Function Induction valueUpregulated genes CYBB Cytochrome b-245, Antioxidant 2.26 0.051 betapolypeptide (chronic granulomatous disease) DUSP1 Dual specificityOxidative stress 2.68 0.023 phosphatase 1 responsive gene GSR Glutationereductase Oxidative stress 2.34 0.016 responsive gene UCP2 Uncouplingprotein Superoxide 3.54 0.044 2(mitochondrial, metabolism protoncattier) VIMP Selenoprotein S Oxidative stress 3.62 0.022 responsivegene Downregulated genes ATOX1 ATX1 antioxidant ROS metabolism −2.830.048 protein CCL5 Chemokine (C-C Oxidative stress −2.28 0.018 motif)ligand 5 responsive gene EPX Eosinophil Antioxidant −2.43 0.046peroxidase MPV17 MpV17 mitochondrial ROS metabolism −3.51 0.04 innermembrane protein PRNP Prion protein Oxidative stress −2.85 0.047responsive gene SOD3 Superoxide Antioxidant −3.28 0.035 dismutase 3,extracellular Positive values indicates up-regulation; negative values,down-regulation.

The expression of five genes, including CYBB, DUSP, GSR, UCP2 and VIMP,were significantly increased, whereas those of six genes, includingATOX1, CCL5, EPX, MPV17, PRNP and SODS, were down-regulated (FIG. 2).Among these, VIMP and MPV17 were most highly up- and down-regulated,respectively.

Finally, as noted above, monocyte levels of treated dogs remained in thenormal range but were significantly increased. Using plasma obtainedfrom the subjects, the level of IFN-γ was found to be increased byapproximately 2-fold during the second phase of the study (13.17±2.66vs. 26.03±1.94 ng/ml). IL-2 was detected in dog plasma before dietarysupplementation (1.12±0.24 ng/ml), but during the second phase, theconcentration decreased to below the minimum reliable detectable dose(0.4 ng/ml). During the first phase of the study, the concentration ofIL-10 was below the lowest detectable level for this cytokine ELISA(0.12 ng/ml), but significant elevation was detected in the second phase(0.63±0.04 ng/ml) (Table 7). The results are depicted in Table 7:

TABLE 7 Cytokine levels in blood plasma. Concentration in Concentrationin plasma, ng/ml plasma, ng/ml Cytokine First phase Second phase IFN-γ13.17 ± 2.66 26.03 ± 1.94 IL-2  1.12 ± 0.24 BDL IL-10 BDL  0.63 ± 0.04Plasma was obtained at the step of lymphocyte isolation. Aftercentrifugation of diluted blood at 340×g for 30 min in a Histopaquegradient, the supernatant (plasma) was collected and frozen at −80° C.ELISA assays were performed as described in the text. The changes in allthree parameters are statistically significant using a parametric pairedt-test for changes from the first phase of the study to the second(p<0.001). The values for BDL (below detectable level) wereconservatively assumed to be 0.1 ng/ml, which is the value specified inthe manuals for each cytokine kit. Additionally, tests for normalityindicate insufficient evidence to assume the changes are not normallydistributed (all p=0.200 for the Kolmogorov-Smirnov test; p>0.15 for theShapiro-Wilk test).

Chemicals and Reagents

The sources of the chemopreventive agents used in this study are listedin Table 1. Prior to conducting the work, all agents were assessed forpurity using LC-UV-MS with a Shimadzu (Kyoto, Japan) IT-ToF highresolution hybrid mass spectrometer equipped with reversed phase HPLC,electrospray and an in-line UV absorbance array detector (Table 1).HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich(St. Louis, Mo.) or Thermo-Fisher (Waltham, Mass.). Distilled water,prepared by a Milli-Q water purification system from Millipore(Milsheim, France) was used throughout the study. Endonuclease III fromE. coli, recombinant, Histopaque-1077, Hank's balanced salt solution(HBSS), dimethylsulfoxide (DMSO), phosphate buffered saline (PBS), andhydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich. Trizolreagent was purchased from Invitrogen™ (Life Technologies, Foster City,Calif.). SYBR® Gold nucleic acid stain was from Life Technologies(Carlsbad, Calif.), Trypan blue solution, 0.4% was purchased from Gibco®(Grand Island, N.Y.). IFN-γ, IL-2 and IL-10 kits were purchased from RayBiotech. (Norcross, Ga.). Comet assay kit for single cell gelelectrophoresis was purchased from Trevigen® (Gaithersburg, Md.). RT²Profiler™ custom made PCR Array: CAPF12480 related to dog oxidativestress was purchased from QIAGEN-Frederick (SABiosciences, Valencia,Calif.) (Table 5). All other chemicals were of analytical grade.

Canine Subjects

Prior to the initiation of dietary experiments, study protocols werereviewed and approved by the University of Hawaii Institutional AnimalCare and Use Committee (IACUC). The methods were carried out inaccordance with the approved guidelines. At each stage of the study, thedogs were examined by the same veterinarian (JALA).

Dogs were client-owned and participated at the owner's consent. Each dogremained on their individual, normal diet for the duration of the study;no special diet was introduced except for that of the biscuit. Inclusioncriteria were as follows: Greater than 1 year of age; in the weightrange of 50-100 pounds; essentially healthy. Any dog not meeting theinclusion criteria was excluded from the study. The eight studyparticipants ranged in age from 2-12 y (median: 6 y), and in weight from49.4-72.4 lbs (median: 60.4 lbs; 27.45 kg). The participants were of thefollowing breeds: Golden retriever, Border collie mix, NewFinland/English setter mix, Golden retriever/Labrador mix,Labrador/Australian shepherd mix, German shepherd/Labrador mix,Labrador, and Labrador mix.

Dog Biscuit Production

The following assumptions were made in the design phase of the study.First of all, in the more typical situation of converting an animal dose(mg/kg) to a human-equivalent dose (mg/kg), one approach is to dividethe animal dose by a factor of 1.8⁶². Here, being presented with dosestaken by human beings, our goal was to determine a canine dose byextrapolation. For example, a value of 1 g/day/person was selected forresveratrol, or around 16 mg/kg body weight. Applying the conversionfactor of 1.8, the canine dose becomes 29 mg/kg, and assuming a bodyweight of 20-30 kg, the daily dosage would be 580-870 mg. These are veryrudimentary conversions, and the safety profiles of the test agents areextremely broad, but weighing on the side of being conservative, a dailydose of 500 mg was selected. A similar approach was used for selectingthe dosages for the remaining chemopreventive agents used in this study.

For the formulation of dog biscuits, the base mix contained wheat flour(0.62 kg), corn meal (0.17 kg), eight beef bouillon cubes (4.136 geach), and water (225 ml). Dry ingredients were weighed out and added toa planetary mixer and mixed for three minutes. Water was added dropwiseover 75 seconds. The mixture was then baked for 60 min at 60° C.yielding a total batch weight of 0.716 kg. The completed base mix wascooled, packaged in plastic bags, and refrigerated. The mixture ofchemopreventive compounds was slugged on a Carver F press. The granulesresulting from gently hand grinding in a mortar and pestle were thenmechanically blended with core material and excipients, and checked forflow ability in a full size hopper. The granules were then mixed with250 g of core base mix and excipients then run through the F pressagain. This required agitation, but the resulting biscuits wereconsistent in weight ranging from 1.4 to 1.5 g. The samples were checkedfor compatibility on the Carver press at 10,000 psi with a 1 min dwelltime. As a result, the following cancer chemopreventive agents wereincluded in the mixture (mg per one biscuit): resveratrol, 250; ellagicacid, 63; genistein, 125; curcumin, 250; quercetin, 250. Placebobiscuits did not contain the chemopreventive agents.

Serum and Plasma Preparation and Complete Blood Counts

Whole blood (6 ml) was collected from each canine subject at the end ofthe first and second phases of the study. Blood was obtained byvenipuncture from either the cephalic or jugular vein (typically with an18-20 gauge needle attached to a 10 ml syringe). Manual restraint andaseptic techniques were used. The blood from each dog was placed inlabelled collection tubes (2 ml in lavender EDTA collection tubes, 1 mlin green lithium heparin collection tubes, and 3 ml in red siliconecoated tubes). The lavender tubes were inverted and used to run CompleteBlood Counts (CBC) and for isolation of lymphocytes, the green tubeswere inverted and used to run Comprehensive Chemistry Blood Profiles(CCBP), and the blood within the red tubes were allowed to clot, andthen centrifuged for 15 min at 3400 rpm to obtain serum. The serum wasfrozen prior to analysis by LC-MS/MS. The CBC and CCBPs were run onAbaxis™ VetScan machines, particularly the VS2 and HM5.

LC-MS and LC-MS/MS

Liquid-liquid extraction was used to prepare dog serum for massspectrometric analysis. Briefly, 300 μl of serum was mixed with 900 μlof ice-cold acetonitrile. Each sample was vortex mixed for 20 sec andthen centrifuged for 10 min at 13,000×g. The supernatant was removed andevaporated to dryness under a stream of nitrogen, and each extract wasreconstituted in 100 μl of acetonitrile/water (20:80, v/v) prior toanalysis using LC-MS/MS.

Analyses of resveratrol and its metabolites, quercetin and genisteinwere carried out using a Shimadzu IT-ToF mass spectrometer for purityconfirmation and a Shimadzu LCMS-8050 triple quadrupole massspectrometer for quantitation. Both instruments were equipped withnegative ion electrospray and interfaced to Shimadzu HPLC systems(Prominence AR or Nexera, respectively). A Waters (Milford, Mass.)XBridge C₁₈ column (2.1×100 mm; 2.5 μm) was used for chromatographicseparations. The solvent system consisted of a 5-min linear gradientfrom 10% to 90% acetonitrile in water at a flow rate of 400 μl/min. Theinjection volume was 10 μl.

For quantitative analysis, selected reaction monitoring (SRM) was usedwith collision-induced dissociation and argon as the collision gas.Deprotonated molecules of each analyte were used as precursor ions, andthe SRM transitions of the two most abundant product ions were selectedas quantifiers and qualifiers, respectively. In some cases, only one SRMtransition (quantifier) was used due to the low abundances of otherfragment ions. For resveratrol, the SRM transitions were m/z 227 to 143and m/z 227 to 185. The SRM transitions of m/z 403 to 227 and m/z 403 to143 were used for resveratrol glucuronide, and m/z 307 to 227 and m/z307 to 143 were used for resveratrol sulfate. The SRM transitions forgenistein were m/z 269 to 89 and m/z 269 to 187, and the SRM transitionsfor genistein glucuronide and genistein sulfate were m/z 445 to 269 andm/z 349 to 269, respectively. Quercetin was measured using the SRMtransitions of m/z 301 to 151 and m/z 301 to 179, and its glucuronideand sulfate metabolites were monitored using the transitions of m/z 477to 301 and m/z 381 to 301, respectively. Synthetic isopentyl naringeninwas used as an internal standard (SRM transition m/z 341 to 119).Although SRM transitions for curcumin (m/z 367 to 134 and m/z 367 to217), curcumin glucuronide (m/z 543 to 367), curcumin sulfate (m/z 447to 367), and ellagic acid (m/z 301 to 229) were monitored, no signalswere obtained for these compounds in the study samples.

Lymphocyte Isolation

For the isolation of canine lymphocytes, the procedure of Strasser etal.⁶³ was followed. Two milliliters of blood in lavender EDTA collectiontubes were placed on ice and delivered to the research laboratory. ColdHistopaque-1077 (2 ml; Sigma-Aldrich, St. Louis, Mo.) solution wascarefully place in 15 ml conical centrifugal polypropylene tubes (Bectonand Dickinson). Blood was diluted with sterile PBS (1:1) at roomtemperature, layered carefully onto the Histopaque-1077, and centrifugedat 340×g for 30 min using a swinging-bucket rotor. Centrifugation wasterminated without applying a brake. The supernatant (plasma) was frozenat −80° C., and the opaque bands at the interface between plasma andHistopaque-1077 containing lymphocytes were collected carefully with asiliconized Pasteur pipet and transferred to separate conicalcentrifugal tubes. Lymphocytes were washed with 10 ml of Hank's balancedsalt solution (HBSS) (two-times; 10 min at room temperature; 300×g).Cells were counted and frozen as aliquots immediately using preservationmedia (9 mL FBS mixed with 1 ml sterile dimethyl sulfoxide) atapproximately −1° C./min in an isopropyl freezing container at −80° C.before storage in liquid nitrogen. The average yield was 10⁶ lymphocytesfrom 1 ml of dog blood. Lymphocytes were distinguished from monocytesand neutrophils by visual inspection. Contamination did not exceed 3%.

In addition, lymphocytes were distinguished from monocytes andneutrophils by microscopic inspection after Diff-Quik staining of fixedcell smears. The nucleus of the lymphocyte appears as a dense formationthat is oval-round and deep purple. Cytoplasm is clear, with nogranules, and only sparse vacuoles. The monocyte nucleus is horse-shoeshaped and not as dense; it is pale purple, lacy and spongy. Thecytoplasm appears grey with numerous vacuoles. The nuclei of neutrophilshave several lobes, and these cells do not layer above Histopaque-1077due to their density. Based on this visual inspection, contamination oflymphocytes with other cell types was found to be less than 3%.

LC-MS and LC-MS/MS. Liquid-liquid extraction was used to prepare dogserum for mass spectrometric.

Cell Viability

Cell viability was determined using the Trypan blue exclusion test.Trypan blue stock solution (0.4%; 10 μl) was added to isolatedlymphocytes (90 μl) and immediately loaded onto a hemocytometer. Thenumber of blue stained cells and the number of total cells wasdetermined by visual inspection. Viability was found to be 90.2±8.3%.Since lymphocytes are readily assessable and play important roles inboth the physiological and pathological processes, it is reasonable toregard them as a useful indicator for oxidative DNA damage⁶⁴, inhumans⁶⁵ as well as canines⁶⁶ and other species¹⁷.

Cytokine Measurements in Plasma

Following the instructions provided by the manufacturer, levels ofIFN-γ, IL-2 and IL-10 in dog plasma were determined with ELISA kitspurchased from Ray Biotech (Norcross, Ga.). The ELISA kits were designedfor in vitro quantitative measurement of samples in serum, plasma, cellculture supernatant. The assays employ antibodies specific for caninecytokines. Assays were validated for each measurement using a set ofcalibration standards.

Comet Assay

The extent of DNA damage in peripheral blood lymphocytes was measured bysingle-cell gel electrophoresis (the alkaline comet assay)^(17,67,68).Cells embedded in agarose on a microscope slide are lysed with detergentand high salt to form nucleoids containing supercoiled loops of DNAlinked to the nuclear matrix. DNA was allowed to unwind under alkalineconditions. Breaks in the DNA molecule disrupt its complex supercoilingallowing free DNA loops to migrate towards the anode duringelectrophoresis. DNA damage to the cells can be thus visualized as“comets” (FIG. 3). Two measures of DNA damage in lymphocytes wereevaluated: endogenous DNA damage (DNA damage without ex vivo H₂O₂exposure) and H₂O₂-inducible DNA damage. Analysis was performedaccording to the method of Singh et al.⁶⁸ and Tice et al.⁶⁹ with somemodifications⁷⁰. A Trevigen® Comet assay kit (Gaithersburg, Md.) wasused for single cell gel electrophoresis. Cells were immobilized in abed of low melting agarose on a Trevigen CometSlide™. A cell suspensioncontaining 1×10⁵ cells per ml was combined with 500 μl of LMAgaroseproviding agarose in a ratio 1:10 for optimal results when spread at 50μl of mixture per slide well. Slides were covered with a glasscover-slip and left at 4° C. in the dark for 10 min. Increasing gellingtime to 30 min improved adherence of the samples. Slides were placed fora minimum of one hour in lysis solution (the buffer formulation isproprietary) to remove membranes, cytoplasm and nuclear proteins. TheCometSlide™ was then immersed in alkaline unwinding solution for 20 minat room temperature or 1 h at 4° C. in the dark. Horizontalelectrophoresis was conducted at 21 volts for 30 min at 4° C. Afterneutralizing, the gels were soaked in 70% ethanol for 5 min and dried at37° C. for 10-15 min. Gels were treated with 100 μl SYBR® Gold nucleicacid stain (Life Technologies) and viewed by fluorescent microscopyusing a Leica TCS SPE confocal microscope (Leica, UK) at Abs/Em 496/540nm. Slides were examined at 400× magnification using image analysissoftware (CometScore from TriTek Corp., Sumerduck, Va.).

From each replicate slide, 50 nuclei were scored and the percentage oftail DNA intensity was used to evaluate the extent of DNA migration anddamage⁶⁶. Fluorescently stained nucleoids from each gel were assessedand classified according to the relative intensity of fluorescence inthe tail undamaged) (FIG. 3). Each cell was visually scored according tothe following criteria: no damage (type 0), mild to moderate damage(type 1 and 2), and extensive DNA damage (type 3 and 4). Under the assayconditions used in this experiment, the intensity of comet tailsreflects electrophoretic migration of DNA resulting from strand breaks.This parameter was used as arbitrary units. Tail length tends toincrease rapidly with dose at low levels of damage, but soon reaches itsmaximum. Tail moment units were not used, since it combines theinformation of tail length and tail intensity, but suffers from lack oflinearity⁶⁶. To detect specifically oxidized pyrimidines in DNA,recombinant bacterial DNA repair enzyme, endonuclease III from E. coli,was used⁶⁵. This provides a specific and sensitive measure of oxidativeDNA damage⁶⁵. The slides were washed three-times after lysis for 5 minat 4° C. with buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mlBSA, pH 8). After blotting dry with tissue paper, the gels wereincubated for 45 min at 37° C. with either 50 μl buffer or endonucleaseIII in buffer (1 μs protein/mL)⁶⁴. All samples were analysed induplicate. Levels of endogenous oxidized pyrimidines demonstrated asimilar pattern as endogenous DNA damage, i.e., no significantdifference between first and second phases (FIG. 1).

DNA Damage Inflicted by H₂O₂

In the next set of experiments, hydrogen peroxide was employed as anoxidant. It has been shown that hydrogen peroxide causes adose-dependent increase in DNA strand breaks in human and doglymphocytes^(66,71). Lymphocytes were thawed, mixed with PBS,centrifuged at 200×g for 3 min at 4° C., and resuspended in PBS at 2×10⁶cells/ml. For H₂O₂ treatment, 10 μl of 1 mM H₂O₂ were added to 90 μl ofcells in PBS. After 5 minutes on ice, the cells were collected bycentrifugation at 300×g for 10 min and analysed by single cell gelelectrophoresis.

Gene Expression Analysis

To explore the potential mechanism underlying the protection effect ofthe dietary supplement on lymphocytes damage caused by H₂O₂, a cDNAmicroarray was employed. For this analysis, RNA was isolated from doglymphocytes and reverse transcribed to cDNA. The same amount of cDNAwith RT² qPCR master mix (25 μl) was placed in each well and subjectedto real-time PCR reaction with the SYBR Green detection system. Usingthe ΔΔCt method changes in gene expression between the two phases of thestudy employing Web-based software from SABiosciences (RT 2 Profiler PCRarray data analysis) were analyzed.

The procedure follows. Total RNA was extracted from 1-2×10⁶ lymphocytesusing Trizol reagent (Invitrogen™) according to the method ofChomczynski and Sacchi⁷². Isolated RNA was dissolved in RNase-free waterand the quality and quantity were measured using a BioSpec-nanospectrophotometer (ShimadzuBiotech). cDNA was generated from total RNAby reverse transcription using a RT² First strand kit from Qiagen on anABI 7300 thermocycler (Applied Biosystems Inc.). cDNA was applied to aRT² profiler custom made PCR Array related to dog oxidative stress(Table 5) following the manufacturer's instructions (QIAGEN-Frederick,SABiosciences). Samples were run in triplicate to ensure amplificationintegrity. Expression levels were analysed with each sample^(73,74).Fold-changes of genes were determined and visualized as heatmaps (red:up-regulation, and green: down-regulation) (FIG. 2).

Statistical Analysis

Results are presented as means±SE. Data representing the various groupswere compared using the Student's t-test and the level of P<0.05 wasconsidered as significantly different. Cytokine data were subjected tothe Kolmogorov-Smirnov and Shapiro-Wilk tests of normality. Otherstatistical considerations are described in the text or tables.

CONCLUSION

As explained above, in order to assess the general health of theanimals, comprehensive blood profiles were examined during the first andsecond phases of the study. No changes of veterinary concern wereobserved, but the population of monocytes was found to be increasedafter the dogs were given the dietary supplement for the treatmentperiod. Previously, it has been shown in dog studies that monocytes andmacrophages are the first cell types responding to neoplastic stimulus,acting through the synthesis of IFN-γ and interleukins⁵⁶. Resveratrol,one of the most studied ingredients in our supplementation, decreasesoxidative burst capacity and changes cytokine production in dogs⁵⁷.Thus, as described above, the plasma concentration of select cytokineswere assessed, and modulation was observed.

The cytokine levels in this study were similar to other studies usingELISA methodology⁵⁸. At this point in time, interpretation of the datais simply based on the observed changes. Interestingly, however, IL-10,elevated in this study, is an anti-inflammatory cytokine that decreasesthe production of pro-inflammatory cytokines, such as TNF-α, IL-1β, andIL-6²². IFN-γ, the concentration of which was possibly increased by CYBBup-regulation^(59,60) (Table 6), affects numerous therapeutic targetswith the potential of producing significant benefits. Using moresensitive methodology, reduction of IL-2 concentrations has beenconsidered significant⁶¹, and here diminution to a level below the limitof detection is reported. Taking into consideration the potentialimportance of immunomodulation in disease prevention¹⁸, based on thesepreliminary findings, additional studies would be of interest. Since itis likely that immunomodulatory mechanisms are distinct from oxidativemechanisms protecting DNA from damage, the implications of the describedstudy are multifaceted.

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Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein, as well as textappearing in the figures and sequence listing, are hereby incorporatedby reference in their entirety for all purposes to the same extent as ifeach were so individually denoted.

1-46. (canceled)
 47. A dog biscuit comprising a therapeuticallyeffective amount of a combination comprising at least three activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts of anyof the foregoing, and mixtures of any of the foregoing sufficient toincrease production of IL-10 in a dog after once-a-day or twice-a-dayadministration of the dog biscuit to the dog.
 48. The dog biscuit ofclaim 47, wherein resveratrol comprises from about 1% to about 80% ofthe combination by weight, ellagic acid comprises from about 0.25% toabout 20% of the combination by weight, genistein comprises from about0.5% to about 40% of the combination by weight, curcumin comprises fromabout 1% to about 80% of the combination by weight; and quercetincomprises from about 1% to about 80% of the combination by weight. 49.The dog biscuit of claim 47, wherein the dog biscuit comprises fromabout 0.05 mg to about 2000 mg of resveratrol.
 50. The dog biscuit ofclaim 47, wherein the dog biscuit comprises from about 0.05 mg to about1000 mg of ellagic acid.
 51. The dog biscuit of claim 47, wherein thedog biscuit comprises from about 0.05 mg to about 1000 mg genistein. 52.The dog biscuit of claim 47, wherein the dog biscuit comprises fromabout 0.05 mg to about 2000 mg curcumin.
 53. The dog biscuit of claim47, wherein the dog biscuit comprises from about 0.05 mg to about 2000mg quercetin.
 54. The dog biscuit of claim 47, wherein the dog biscuitcomprises resveratrol and at least two active agents selected from thegroup consisting of genistein, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts, and mixtures of any of the foregoing, andprovides a serum level of resveratrol of from about 3.86 ng/ml to about154 μg/ml in the dog after said administration.
 55. The dog biscuit ofclaim 47, wherein the dog biscuit comprises resveratrol and at least twoactive agents selected from the group consisting of genistein, ellagicacid, curcumin, quercetin, prodrugs, metabolites, salts, and mixtures ofany of the foregoing, and provides a serum level ofresveratrol-3-O-glucuronide of from about 12.9 ng/ml to about 514 μg/mlin the dog after said administration.
 56. The dog biscuit of claim 47,wherein the dog biscuit comprises resveratrol and at least two activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, curcumin, quercetin, prodrugs, metabolites, salts, andmixtures of any of the foregoing to provide a serum level oftrans-resveratrol-4′-sulfate of from about 8.9 ng/ml to about 355 μg/mlin the dog after said administration.
 57. The dog biscuit of claim 47,wherein the dog biscuit comprises resveratrol and at least two activeagents selected from the group consisting of genistein, ellagic acid,curcumin, quercetin, prodrugs, metabolites, salts, and mixtures of anyof the foregoing and provides a serum level oftrans-resveratrol-3-sulfate of from about 84.9 ng/ml to about 3397 μg/mlin the dog after said administration.
 58. The dog biscuit of claim 47,wherein the dog biscuit comprises resveratrol and at least two activeagents selected from the group consisting of genistein, ellagic acid,curcumin, quercetin, prodrugs, metabolites, salts, and mixtures of anyof the foregoing and provides a serum level of cis-resveratrol-3-sulfateof from about 2.7 ng/ml to about 108 μg/ml in the dog after saidadministration.
 59. The dog biscuit of claim 47, wherein the dog biscuitcomprises resveratrol and at least two active agents selected from thegroup consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts, and mixtures of any of theforegoing and provides a serum level of resveratrol sulfate total offrom about 36.7 ng/ml to about 1467 μg/ml in the dog after saidadministration.
 60. The dog biscuit of claim 47, wherein the dog biscuitcomprises genistein and at least two active agents selected from thegroup consisting of resveratrol, ellagic acid, curcumin, quercetin,prodrugs, metabolites, salts, and mixtures of any of the foregoing andprovides a serum level of genistein-4′-glucuronide of from about 14μg/ml to about 14167 μg/ml in the dog after said administration.
 61. Thedog biscuit of claim 47, wherein the dog biscuit comprises genistein andat least two active agents selected from the group consisting ofresveratrol, ellagic acid, curcumin, quercetin, prodrugs, metabolites,salts, and mixtures of any of the foregoing and provides a serum levelof genistein-4′-glucuronide of from about 1.7 μg/ml to about 1700 μg/mlin the dog after said administration.
 62. The dog biscuit of claim 47,wherein the dog biscuit comprises genistein and at least two activeagents selected from the group consisting of resveratrol, ellagic acid,curcumin, quercetin, prodrugs, metabolites, salts, and mixtures of anyof the foregoing to provide a serum level of genistein sulfate of fromabout 54 μg/ml to about 53836 μg/ml.
 63. The dog biscuit of claim 47,wherein the dog biscuit comprises curcumin and at least two activeagents selected from the group consisting of resveratrol, genistein,ellagic acid, quercetin, prodrugs, metabolites, salts, and mixtures ofany of the foregoing and provides a serum level of curcumin glucuronideof from about 6.25 ng/ml to about 250 μg/ml in the dog after saidadministration.
 64. The dog biscuit of claim 47, wherein the dog biscuitcomprises quercetin and at least two active agents selected from thegroup consisting of resveratrol, genistein, ellagic acid, curcumin,prodrugs, metabolites, salts, and mixtures of any of the foregoing andprovides a mean serum level of quercetin of from about 0.2 ng/ml toabout 8000 μg/ml.
 65. The dog biscuit of claim 47, wherein the dogbiscuit comprises resveratrol, genistein, curcumin, quercetin and, afteradministration to a dog, provides (i) a serum level of resveratrol offrom about 3.86 ng/ml to about 154 μg/ml; (ii) a serum level ofresveratrol-3-O-glucuronide of from about 12.9 ng/ml to about 514 μg/ml;(iii) a serum level of trans-resveratrol-4′-sulfate of from about 8.9ng/ml to about 355 μg/ml; (iv) a serum level oftrans-Resveratrol-3-sulfate of from about 84.9 ng/ml to about 3397μg/ml; (v) a serum level of cis-resveratrol-3-sulfate of from about 2.7ng/ml to about 108 μg/ml; (vi) a serum level of resveratrol sulfatetotal of from about 36.7 ng/ml to about 1467 μg/ml; (vii) a serum levelof genistein-4′-glucuronide of from about 14 μg/ml to about 14167 μg/ml;(viii) a serum level of genistein-4′-glucuronide of from about 1.7 μg/mlto about 1700 μg/ml; (ix) a serum level of genistein sulfate of fromabout 54 μg/ml to about 53836 μg/ml; (x) a mean serum level of curcuminglucuronide of from about 6.25 ng/ml to about 250 μg/ml; and (xi) aserum level of quercetin of from about 0.2 ng/ml to about 8000 μg/ml.66. A dog biscuit comprising a therapeutically effective amount of acombination comprising at least three active agents selected from thegroup consisting of resveratrol, genistein, ellagic acid, curcumin,quercetin, prodrugs, metabolites, salts of any of the foregoing, andmixtures of any of the foregoing sufficient to increase a number ofmonocytes and macrophages in a dog after once-a-day or twice-a-dayadministration of the dog biscuit to the dog.